ABSTRACT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective loss of motor neurons. Stress activated protein kinases (SAPK) have been suggested to play a role in the pathogenesis of ALS. We studied the relevance of p38 MAPK for motor neuron degeneration in the mutant SOD1 mouse. Increased levels of phospho-p38 MAPK were present in the motor neurons and microglia of the ventral spinal cord. The p38 MAPK-inhibitor, SB203580, completely inhibited mutant SOD1-induced apoptosis of motor neurons and blocked LPS-induced activation of microglia. Semapimod, a p38 MAPK inhibitor suitable for clinical use, prolonged survival of mutant SOD1 mice to a limited extent, but largely protected motor neurons and proximal axons from mutant SOD1-induced degeneration. Our data confirm the abnormal activation of p38 MAPK in mutant SOD1 mice and the involvement of p38 MAPK in mutant SOD1-induced motor neuron death. We demonstrate the effect of p38 MAPK inhibition on survival of mutant SOD1 mice and reveal a dissociation between the effect on survival of motor neurons and that on survival of the animal, the latter likely depending on the integrity of the entire motor axon.

5.1). To quantify motor neuron survival in vivo, hematoxylin–eosinstaining was performed in order to count the number of neurons inthe ventral horn after semapimod treatment (5 slides per animalwere quantified, n=3 per group). Motor neurons were defined aslarge, polymorphic cells with a prominent nucleolus and an area ofmore than 50 μm2in the nucleolar plane. Perfused ventral lumbarspinal roots were dissected and embedded in glutaraldehyde. Afterstaining with toluidine blue, ultra thin sections were cut and thenumber of axons and the surface of the ventral root were assessedusing Lucia imaging software (version 4.60, Laboratory Imaging,Praha, Czech Republic).Cell culturesFor microglial cultures, total brains were dissected fromnewborn Wistar rat pups. Blood vessels and meninges wereremoved. Tissues were collected and washed four times withcooled PBS, homogenized mechanically without enzymes and cutinto small pieces and filtered through a 70 μm cell strainer(Falcon, Becton Dickinson Labware, NJ). PBS was replaced intwo centrifugation steps with low-glucose solution and cells wereresuspended in medium (Dulbecco's Modified Eagle Medium(DMEM; Gibco), supplemented with low glucose and glutamine(GlutaMAXTM-I, Gibco), 10% fetal calf serum (Gibco), penicillin(100 U/ml) and streptomycin (100 μg/ml)). Cells were plated onpoly-L-ornithine coated culture flasks (one brain per flask) andmaintained in a humidified incubator at 37 °C and 7% CO2. Themedium was replaced once weekly. This procedure yielded amixed culture, from which, 2 weeks after preparation, microglialcells were harvested by gently shaking the culture flask andcollecting the medium. Cells were seeded in medium withoutantibiotics on 6-well plates at a density of 50000 cells/cm2forWestern blot analysis and at a density of 10000 cells/cm2on twochambered Lab-TekTMslides (Nalge Nunc) for immunocyto-chemistry. For NO concentration experiments, a BV-2 microglialcell line was used.Purity of microglial cultures was confirmed by demonstratingthat specific staining with the microglial marker OX-42 waspresent in approximately 95–99% of all cultured cells (data notshown).Primary motor neuron cultures were prepared as previouslydescribed (Van Den Bosch et al., 2002a). Briefly, spinal cords weredissected from E13 embryos and collected in Hanks' Balanced SaltSolution (HBSS; Gibco). Ventral cords were cut into pieces ofabout 1 mm and digested for 15 min at 37 °C in 0.05% trypsin inHBSS. The solution was then replaced with culture medium (L15,Sigma) supplemented with 3.6 mg/ml glucose, 20 nM progester-one, 5 mg/ml insulin, 0.1 mM putrescine, 0.1 mg/ml conalbumin,30 nM sodium selenite, 100 U/ml penicillin, 100 mg/ml strep-tomycin and 2% horse serum) containing 0.4% BSA (Serva) and20 μg/ml DNase and the tissue was further dissociated bytrituration. The resulting single cell suspension was layered on a6.8% (w/v in L15) metrizamide cushion (one spinal cord per tube),and centrifuged at 500×g for 15 min. This resulted in a sharp band(fraction F1) on top of the metrizamide cushion and a pellet(fraction F2). To remove debris, both fractions were resuspended inculture medium and centrifuged for 20 min at 75×g on a 4% BSAcushion. After genotyping the embryo, 30,000 motor neurons ofthe F1 fraction were seeded on a glial feeder layer. Glial feederlayers were prepared by plating F2 cells in 35 mm dishes. After 4weeks in vitro, cell division was halted by exposure to 10−5Mcytosine arabinoside. After seeding of the motor neurons on thefeeder layer, cultures were maintained in culture mediumsupplemented with 0.2% sodium bicarbonate. Culture mediumwas replaced twice weekly. Exposure experiments with cyclospor-ine A and the p38 MAPK inhibitors (SB203580 and semapimod)were also performed in culture medium supplemented with 0.2%sodium bicarbonate.Exposure experiments and assessment of neuronal survivalAt 8 days in vitro, motor neuron co-cultures derived from eitherSOD1WTembryos or mutant SOD1G93Aembryos were exposed to5 μM cyclosporine A (Sigma) in culture medium for 24 h. Neuronalsurvival was quantified by direct counting of unfixed neuronsunder phase contrast optics at 100×. Neurons were counted imme-diately before and 24 h after cyclosporine A exposure within anidentified region of 1 cm2(10% of the total area of the dish) using agrid. The percentage neuronal survival was determined as the ratioof the number of neurons after 24 h of cyclosporine A exposure tothe baseline number prior to treatment and this ratio was thennormalized to controls performed in the same conditions. Neuronswere counted by an observer blinded to the treatment protocol.Primary microglial (Western blot) and BV-2 microglial cultures(NO concentration) were stimulated for different periods of time(0 h, 3 h and 6 h) with 1 ng/ml lipopolysaccharide (LPS;Calbiochem). Where mentioned, SB203580 (10 μM or 50 μM;Calbiochem) or zVAD-fmk (100 μM; Calbiochem) was added tothe medium of motor neurons and microglia 30 min beforeexposure to cyclosporine A or LPS, respectively, and remainedpresent during the exposure experiments. NO concentration wasmeasured using Griess reagent (Sigma) as described previously(Reis et al., 2006).StatisticsData are presented as the mean±SEM. Statistical comparisonswere made by a one-way ANOVA and a Mann–Whitney U test forsurvival analysis, using StatsDirect 1.8.6.ResultsAge-dependent activation of p38 MAPK, but not JNK,in SOD1G93Amotor neurons and microglial cellsTo quantitatively describe the expression levels of p38 MAPKand its active, phosphorylated form, Western blot analysis of totalspinal cord homogenates of mutant SOD1G93Aand SOD1WTmiceat 80 days (presymptomatic), 120 days (early symptomatic) and130–140 days (end stage of the disease) was performed.Upregulation of phospho-p38 MAPK in mutant SOD1G93Aspinalcord as compared to SOD1WTbecame evident early in the disease(Fig. 1A). Total amount of p38 MAPK was upregulated at endstage of the disease only, and only to a limited extent (Fig. 1A).Western blot of mutant SOD1G93Aand SOD1WTanimals at theages mentioned showed no significant upregulation of phos-phorylated p46 and p54 JNK (Fig. 1B). Western blot analysis ofventral and dorsal parts of the spinal cord separately demonstratedthat the abnormal phospho-p38 MAPK activation was mainlyaccounted for by the upregulation in the ventral half of the spinalcord (Fig. 1C).3 M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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Double immunostaining experiments of mutant SOD1G93AandSOD1WTspinal cords at the age of 120 days showed that phospho-p38 MAPK was present in both motor neurons and microglia (Fig.2), as has been reported previously (Bendotti et al., 2004). In motorneurons, it was mainly present in perinuclear aggregates. No colo-calization with GFAP was found, making p38 MAPK activation inastrocytes unlikely.p38 MAPK inhibition abolishes mutant SOD1-induceddegeneration of motor neurons in vitro, independently of itsblocking effect on microglial activationAs phospho-p38 MAPK was present in both motor neurons andmicroglia, we studied the effect of the p38 MAPK inhibitorSB203580 on microglial activation and on SOD1G93A-dependentmotor neuron death in vitro.Treatment of microglial cultures with LPS resulted in a time-dependent increase in the level of phospho-p38 MAPK and wasaccompanied by the release of NO, a measure of microglialactivation (Fig. 3A). Treatment of the cultures with SB203580, aspecific inhibitor of p38 MAPK activity (Cuenda et al., 1995),30 min prior to exposure to LPS effectively inhibited this NOrelease (Fig. 3A). In line with the mechanism of action ofSB203580, which interferes with the activity of p38 MAPK, butnot with its phosphorylation, no significant effect was observed onWestern blot (Fig. 3A).The effect of p38 MAPK inhibition on SOD1G93A-induced celldeath was evaluated in motor neurons cultured on a feeder layer ofastroglial cells. This feeder layer consists of nearly 100% GFAP-positive cells, while no CD11b-positive cells are present, indicatingthe absence of microglial cells in the culture (data not shown).SOD1G93A-dependent motor neuron death was induced by cyclo-sporine A as reported before (Fig. 3B) (Krishnan et al., 2006; VanDenBoschetal.,2004).TreatmentofthecultureswithzVAD-fmk,abroad spectrum caspase inhibitor, completely inhibited motor neu-ron death, suggesting its apoptotic nature. SB203580 completelyabolished mutant SOD1G93A-dependent motor neuron death. Thissuggests that mutant SOD1G93A-dependent motor neuron apoptosisis mediated through the p38 MAPK pathway and that the effect ofinhibitors of the p38 MAPK system in vivo can be mediated bothby an effect on microglial cells, but also by a direct anti-apoptoticeffect on motor neurons, as has been suggested before (Raoul et al.,2002, 2005).Effect of inhibition of p38 MAPK in SOD1G93AmiceBecause the in vitro experiments showed that p38 MAPKinhibition had an effect on both microglial activation and onmutant SOD1G93A-dependent apoptosis of motor neurons, weevaluated the effect of p38 MAPK inhibition in vivo. Wetherefore treated mutant SOD1G93Amice with 4 mg/kg ofsemapimod daily from the age of 70 days until end stage of thedisease. One molecular target of semapimod is c-Raf, whichactivates p38 MAPK and JNK through MEK phosphorylation(Lowenberg et al., 2005). This compound is under developmentfor the treatment of a variety of inflammatory diseasescharacterized by p38 MAPK upregulation (Akerlund et al.,1999; Denham et al., 2000; Hommes et al., 2002). Intraperitonealadministration of semapimod blocks intrathecal HIV-1 gp120-induced thermal hyperalgesia and mechanical allodynia (Milliganet al., 2001) and attenuates the development of experimentalautoimmune encephalomyelitis (Zinser et al., 2004) demonstrat-Fig. 1. Phospho-p38 MAPK is upregulated early in the disease. (A) Western blot analysis (upper panel) and quantification (lower panel) of total spinal cordhomogenates stained with an antibody directed against total p38 MAPK (p38 MAPK) and its phosphorylated form (p-p38 MAPK) at different ages:asymptomatic (asympt, 80 days), early symptomatic (early, 120 days) and end stage of disease (late, 130–140 days). Samples of mutant SOD1G93A(mt; whitebars) and SOD1WT(wt; black bars) mice were compared (*p<0.05; n=4 per group). (B) Western blot (upper panel) and quantification (lower panel) of totalspinalcordofmutantSOD1G93A(mt; blackbars)andSOD1WT(wt;whitebars)miceat twodifferent agesstainedforboth formsofphosphorylated JNK(p-JNK):p54 and p46, showing no significant difference between the two genotypes at different ages (p>0.05; n=3 per group). (C) Western blot (upper panel) andquantification (lower panel) of the ventral part (v) and the dorsal part (d) of the spinal cord analyzed separately at the age of 120 days in both SOD1WT(WT) andmutant SOD1G93A(G93A) mice (*p<0.01; n=3 per group).4 M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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ing that systemic administration affects central nervous systempathophysiological mechanisms.To ascertain that semapimod inhibits p38 MAPK in vivo, weperformed Western blotanalysis of spinal cord homogenates of bothtreated and control SOD1G93Amice and showed that semapimodeffectively inhibited p38 MAPK activation in the SOD1G93Aspinalcord (Fig. 4A). Semapimod similarly reduced tau phosphorylationat the Thr231residue (Fig. 4B), which is a known substrate foractivated p38 MAPK (Feijoo et al., 2005; Ferrer, 2004).Immunohistochemical analysis of the spinal cord of both controlmice and semapimod-treated mice confirmed the reduction of p38MAPK phosphorylation found on Western blot (data not shown).We then evaluated the effect of i.p. treatment of semapimodboth on onset of the disease and survival in mutant SOD1G93Amice. Semapimod delayed onset of disease by 5 days, a differencethat did not reach statistical significance (109.8±2.3 days for thesemapimod-treated group (n=5) and 105.1±4.5 days for thecontrol group (n=7); p=0.3). Semapimod prolonged survival by 6days, a small but statistically significant increase of life span with4.5% (142.8±2.0 days for the semapimod-treated group (n=23)and 136.6±1.8 days for the control group (n=21); p=0.048)(Fig. 5).The effect of semapimod treatment on motor neuron survivaland integrity of motor axons in the ventral root in both groups wasdetermined at 120 days. Surprisingly, semapimod rescued motorneurons to a large extent, while no effect was seen on the numberof smaller non-motor neurons (Table 1 and Fig. 6A). Similar towhat we observed on motor neuron cell bodies, semapimod alsoFig. 2. Phospho-p38 MAPK is upregulated in microglia and motor neurons. Immunohistochemical double staining of the spinal cord of a 120 days old mutantSOD1G93A(A, C, E–G) mouse and a SOD1WT(B, D) animal. Phospho-p38 MAPK (blue) expression occurred in the perinuclear aggregates (arrow) of motorneurons (SMI-32, green) of the mutant mouse (A), but not in the wild-type (B). No expression of phospho-p38 MAPK (blue) occurred in the astrocytes (GFAP,green) of the mutant (C) or the wild-type (D) mice. Asterisks indicate stellate cells expressing phospho-p38 MAPK, corresponding to microglial cells. (E–G)High magnitude confocal image of microglial cells in the ventral spinal cord of a mutant SOD1G93Aanimal. Microglia were identified using an antibody directedagainst CD11b (E) and showed clear expression of phospho-p38 MAPK (F). Arrowheads indicate microglial cells. Panel G shows merge of E and F. Scalebar=50 μm.5 M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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protected the proximal axons as evaluated by measuring axonaldensity in the ventral roots although to a lesser extent (Fig. 6B).These findings demonstrate a clear effect of semapimod onSOD1G93A-induced motor neuron apoptosis, reflected by enhancedmotor neuron survival and the protective effect on ventral rootaxons, and an effect on survival, smaller than expected on the basisof the effect observed on motor neuron survival in the spinal cord.We hypothesized that this dissociation is due to a differential effectof p38 MAPK inhibition on the perikaryon and the axon.DiscussionThe present study shows that the loss of motor neurons in ALSisaccompanied by a significant activation of p38 MAPK. Thisactivation becomes apparent at disease onset. The increase ofphospho-p38 MAPK almost exclusively occurs in the ventral partof the spinal cord. In agreement with the study by Veglianese et al.(2006), our data indicate that most of the activation of p38 MAPK isaccounted for by the robust upregulation in the proliferatingFig. 4. Treatment with semapimod inhibits p38 MAPK activation in vivo. (A) Western blot analysis (upper panel) and quantification (lower panel) of total spinalcord homogenates of mutant SOD1G93Amice at the age of 120 days treated with mannitol (C) or semapimod (Smp) showing that semapimod significantlyinhibits p38 MAPK phosphorylation in vivo (*p<0.05). Similar effects were found for phosphorylation of tau (B), which is a downstream target of p38 MAPK(**p<0.01).Fig. 3. Inhibition of p38 MAPK activity tempers microglial activation and protects motor neurons in vitro. (A) Upper panel: Western blot analysis ofhomogenates of a purified microglial culture treated with LPS for different periods of time after 30 min pretreatment with either culture medium (C) or 50 μMSB203580 (SB). LPS treatment results in a significant increase in the levels of phospho-p38 MAPK as early as after 3 h of treatment. In line with its mechanismof action, the inhibition of the activity of p38 MAPK by SB203580 is not reflected by a decreased phosphorylation of the kinase. Lower panel: quantification ofthe production of NO after LPS treatment of BV-2 microglial cells. Pretreatment with SB203580 yields a significant reduction of the NO concentration inresponse to LPS (*p<0.05). (B) Motor neuron survival after addition of 5 μM cyclosporine A (CyA) and after pre-treatment for 30 min with either SB203580(SB) or the pan-caspase inhibitor zVAD-fmk (zVAD). This experiment shows that CyA induces mutant SOD1G93A-dependent cell death that is apoptotic innature as it can be rescued by zVAD-fmk and is also p38 MAPK-dependent (**p<0.01).6 M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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microglia, but unlike that study we did not find astrocytes to expressphospho-p38 MAPK. We also found increased phospho-p38MAPK to be present in motor neurons, in which it occurred inperinuclearly located aggregates as described before (Bendotti etal., 2004). No colocalization of these perinuclear inclusions andSOD1 was found (data not shown); hence the precise significanceof these perinuclear inclusions remains elusive.We did not observe an abnormal activation of the JNK pathwayin the mutant SOD1 mouse, a finding in agreement with Holasek etal. (2005), but in contrast to other reports in which JNK activationwas present in astrocytes and microglia (Migheli et al., 1997;Veglianese et al., 2006).Our in vitro experiments show that inhibitors of p38 MAPK cansuppress microglial activation as has been reported before (Tikka etal., 2001). More importantly, we here demonstrate that inhibition ofp38 MAPK activity can also rescue motor neurons in a paradigm ofcyclosporine A-induced, mutant SOD1G93A-dependent motorneuron death. Motor neuron death in this in vitro model is apoptoticin nature as it was completely blocked by the broad spectrumcaspase inhibitor zVAD-fmk. As SB203580, a selective p38 MAPKinhibitor, completely protected motor neurons as well, mutantSOD1-induced cell death in this in vitro paradigm appears to belargely mediated through p38 MAPK activation. These observa-tions support the existence of a motor neuron-specific p38 MAPK-dependent cell death pathway, as has been suggested by Raoul et al.(2005). These authors described a motor neuron-specific Fas-dependent cell death pathway involving p38 MAPK activation inwhich FasL- and NO-induced motor neuron death in vitro could bealmost completely inhibited by SB203580, a p38 MAPK inhibitor(Raoul et al., 2005). Mutant SOD1 motor neurons were moresensitive to this p38 MAPK-mediated FasL-induced death pathwaythan were wild-type SOD1 motor neurons. Hence, our data confirmthe existence of the motor neuron specific death pathway using acompletely independent and alternate experimental setup.p38 MAPK inhibition thus appeared to inhibit both microglialactivation and motor neuron apoptosis. The latter effect isindependent of the effect on microglia, as no microglial cells arepresent in the motor neuron/astroglial co-cultures in which weobserved the anti-apoptotic effect on motor neurons. This dualeffect of p38 MAPK inhibition explains the apparently contra-dictory hypotheses concerning the mechanism of action underlyingthe effect of minocycline in the mutant SOD1 mice.Encouraged by our in vitro data, we treated mutant SOD1G93Amice with semapimod, a p38 MAPK inhibitor in clinicaldevelopment. This compound was developed as a potent inhibitorof TNF-alpha synthesis in macrophages (Cohen et al., 1996), butthe mechanism of action was found to involve inhibition of p38MAPK and JNK phosphorylation through the inhibition of c-Raf(Lowenberg et al., 2005). To date, the small molecule semapimodhas been used in the treatment of therapy resistant patients withCrohn's disease, in whom the treatment resulted in a decreaseddisease activity and clinical remission (Hommes et al., 2002).Animal studies indicated that the compound has clinical activity inthe treatment of rheumatoid arthritis, auto-immune inflammatorydiseases and adult respiratory distress syndrome (Akerlund et al.,1999; Denham et al., 2000; Martiney et al., 1998).Treatment of mutant SOD1G93Amice with semapimodsignificantly decreased the activation of p38 MAPK in the spinalcord, and reduced tau phosphorylation, indicating that the systemicadministration of the drug resulted in its expected biochemicalactivity in the central nervous system. Semapimod treatmentdelayed disease onset by 4 days and increased survival by 6 days,differences of which only the latter reached statistical significance.As p38 MAPK inhibition was far from complete, larger effectsmay have been obtained with higher doses of the drug. However,the dose used was the highest dose tolerated by the animals(Akerlund et al., 1999). This limited effect contrasts with theobvious effect of semapimod on survival of motor neurons and itsrescuing effect on proximal axons, although the latter was lessimpressive than the former. This dissociation between clinicaloutcome and pathological features suggests that the compoundinhibited motor neuron death efficiently, but was not able to restorethe function of the entire motor unit including axon andneuromuscular junction, in order to affect survival to a largerdegree. This observation is reminiscent of the recent finding thatdeletion of the pro-apoptotic bax gene from the mutant SOD1mouse results in almost complete rescue of the motor neurons inthe ventral horn, even in the final stages of the disease, butincreases life span to only a limited extent (Gould et al., 2006). Theauthors of this report described that in spite of the positive effectson motor neuron survival, denervation of the neuromuscularjunctions was only moderately delayed, rather than rescued.Therefore, the anti-apoptotic effect of inhibition of p38 MAPKactivation described in the present study, most likely rescues themotor neuron perikaryon and to a lesser degree the proximal axon,but that distal denervation progresses, underscoring the notion thatdysfunction of the distal neuromuscular compartment is the majordeterminant of progression and severity in ALS (Fischer et al.,2004; Sagot et al., 1995). Clearly the limitations of in vitro modelsfor the study of the potential therapeutic value of a compoundshould be kept in mind. Moreover, it is cautious to emphasize thatthe predictive value of therapeutic interventions in the mutantSOD1 mouse for human ALS remains to be established. Takentogether, our results suggest that p38 MAPK inhibition may have aTable 1Neuronal survival after semapimod treatment at 120 daysNeuronal area (μm2)<100100–200 >200ControlSemapimodp-value31.5±2.325.8±2.00.0823.4±2.840.3±3.60.0016.9±1.216.4±2.00.0003Fig. 5. Semapimod prolongs survival of mutant SOD1G93Amice moderately.Kaplan–Meier survival curve showing the effect on survival of semapimodtreatment (n=23) compared to control (mannitol; n=21) (p=0.048).7M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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therapeutic benefit in ALS, but that the clinical benefit may besmaller than what may be expected on basis of its positive effect onmotor neuron apoptosis.AcknowledgmentsThis work was supported by grants from the Fund for ScientificResearch Flanders (F.W.O. Vlaanderen), the Stem Cell InstituteLeuven and the University of Leuven. M.D. is a Research Assistantof the Fund for Scientific Research Flanders. This research projectis part of the Interuniversity Attraction Poles program P5/19 of theBelgian Federal Science Policy Office (Molecular Genetics andCell Biology). WR is supported through the E. van Behring Chairfor neurodegenerative and neuromuscular disorders.ReferencesAkerlund, K., Erlandsson Harris, H., Tracey, K.J., Wang, H., Fehniger, T.,Klareskog, L., Andersson, J., Andersson, U., 1999. Anti-inflammatoryeffects of a new tumour necrosis factor-alpha (TNF-alpha) inhibitorFig. 6. Semapimod significantly rescues motor neurons and axons. (A) Left panel: immunohistochemical staining of the ventral spinal cord of mutant SOD1G93Amice aged 120 days treated with vehicle (control) or with the active compound (semapimod). An antibody directed against SMI-32 was used. In control animalsonly few motor neurons were left (asterisks). In semapimod treated animals significantly more motor neurons with normal morphology were preserved(asterisks). Boundary between gray (gm) and white matter (wm) is indicated with the dotted line. Scale bar: 20 μm. Right panel: quantification of the number ofmotor neurons counted in the ventral part of the spinal cord of 3 to 4 different animals per group, showing that the treatment mainly affects motor neurons with alarge cell surface, while no significant effect is seen in smaller neurons (*p<0.005). (B) Left panel: semi-thin sections of the lumbar ventral root of a control (C)and semapimod (smp) treated mouse. Right panel: quantification of the axonal density in both conditions at the age of 120 days (*p<0.05).8 M. Dewil et al. / Neurobiology of Disease xx (2007) xxx–xxxARTICLE IN PRESSPlease cite this article as: Dewil, M., et al., Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1G93A-induced motor neurondeath, Neurobiol. Dis. (2007), doi:10.1016/j.nbd.2006.12.023

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[Show abstract][Hide abstract]ABSTRACT:
Amyotrophic lateral sclerosis (ALS) is an adult onset degenerative disease characterized by the selective progressive death of lower and upper motor neurons. Although its primary cause remains unknown, multiple pathogenic pathways have been identified. The major pathological event in ALS is the degeneration of lower motor neurons (LMNs) and it is thought to be the ultimate cause of death. Thus neuroprotection of LMNs is assumed to be a reasonable target for treating ALS. This assumption guided most of research effort in the last 20 years to develop neuroprotective strategies able to preserve the remaining LMNs. Three major possibilities have been explored and are presented in this review. First, it is thought that LMN survival and degeneration is regulated by neurotransmittory inputs that LMN integrates. Several experimental and imaging studies have show an impairment of multiple neuronal types innervating motor neurons, in particular glycinergic interneurons, glutamatergic input from proprioceptive type Ia fibers, and serotonergic neurons. The abnormalities in glutamatergic input proved especially fruitful since they led to the discovery of riluzole. A second potential neuroprotective strategy would be to modulate neurotrophic input to motor neurons. Indeed, studies of motor neuron development have shown that their survival is governed by neurotrophic input from its muscle target or from surrounding glia. Thus, modulating neurotrophic support to LMN might be a therapeutic strategy, although the multiple clinical trials based on this possibility were up to now unsuccessful. A last possiblity to provide neuroprotection in ALS would be to indirectly modulate the motor neuron survival through action on events occuring outside this cell type, in particular inflammation or abnormal energy metabolism.

[Show abstract][Hide abstract]ABSTRACT:
To evaluate the effects of intrathecal administration p38β antisense oligonucleotide on the development of bone cancer pain rats.
Forty female SD rats weighing 180~220 g were randomly divided into 4 groups (n = 10 each): Group A (control group): intra-tibial injection of 3 μl Hank's solution; group B (model group): intra-tibial injection of 3 μl MADB-106 mammary gland carcinoma cells of rats (4.8 × 10(3)/μl); group C (p38β-SODN 20 μg); group D (p38β-ASODN 20 μg). The model procedures in group C and D were same to those in the group B. From the 14(th) day after operation, p38β-SODN 20 μg and p38β-ASODN 20 μg were respectively intrathecally administrated in group C and D once daily for 6 days whereas normal saline was for group A and B. Mechanical withdrawal threshold and radiant heat threshold of rat hind paws were measured before operation and every other day until 22 d of post-operation. The lumbar 4-6 spinal cord was removed on the 22(nd) day. The expression of spinal p38β protein was determined by Western blot.
No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in control group. During the first 6 days after operation there were obvious differences in radiant heat stimulus between control group between the other groups (P < 0.05); During 14-22 days after operation, mechanical pain threshold and radiant heat threshold between p38β-SODN group and Model group were significantly changed compared with that in control group (P < 0.05). However, the differences were not remarkable between control group and p38β-ASODN group (P > 0.05). The expression of p38β protein in lumbar spinal cord was significantly higher between p38β-SODN group and Model group than that in control group (P < 0.05). There was no significant difference in p38β protein expression between p38β-ASODN group and control group (P > 0.05).
Hyperalgesia induced by bone cancer can be inhibited by intrathecal administration of p38β antisense oligonucleotide, which is achieved by reducing expression of p38β protein.

[Show abstract][Hide abstract]ABSTRACT:
For over a century Drosophila melanogaster, commonly known as the fruit fly, has been instrumental in genetics research and disease modeling. In more recent years, it has been a powerful tool for modeling and studying neurodegenerative diseases, including the devastating and fatal amyotrophic lateral sclerosis (ALS). The success of this model organism in ALS research comes from the availability of tools to manipulate gene/protein expression in a number of desired cell-types, and the subsequent recapitulation of cellular and molecular phenotypic features of the disease. Several Drosophila models have now been developed for studying the roles of ALS-associated genes in disease pathogenesis that allowed us to understand the molecular pathways that lead to motor neuron degeneration in ALS patients. Our primary goal in this review is to highlight the lessons we have learned using Drosophila models pertaining to ALS research.